Regeneration of normal shape, architecture and function of craniofacial tissues due to congenital abnormality, trauma or surgical treatment presents special problems to tissue engineering. Because of the great variations in properties of these tissues, currently available treatment options fall short of adequate care. The availability of customized living tissues engineered in vitro would revolutionize the way we currently treat craniofacial defects. In the 1st grant cycle, we established a tissue engineering approach to craniofacial reconstruction based on anatomically shaped cartilage/bone grafts formed using human stem cells, composite scaffolds and perfusion/loading bioreactors. From these studies, we published 74 peer-reviewed journal articles, 27 of which were coauthored by the two laboratories (Columbia University and Tufts University). For the 2nd grant cycle, we will maintain our focus on the temporomandibular joint (TMJ) and build upon the results of the 1st cycle to make advances in three important areas: (i) Develop composite scaffolds mimicking the internal architectures of craniofacial tissues, (ii) Integrate bioreactors with live imaging to study tissue development without interrupting cultivation, and (iii) Conduct a large animal study of craniofacial reconstruction. The research plan has been designed for impact in two areas: (i) Customized approach to craniofacial reconstruction, and (ii) Quantitative insights into the formation of craniofacial tissues, using an advanced bioreactor-imaging system and a clinically relevant animal model. Our overall hypothesis is that craniofacial grafts can be formed by biophysical regulation of adult human stem cells using a biomimetic scaffold-bioreactor system. We will engineer the TMJ condyle and the TMJ disc, two important and interacting jaw components, with complex architectures and mechanical loading, but with distinct structures. Three specific aims will be pursued: Aim 1 - Develop anatomically shaped scaffolds for engineering TMJ condyle and disc, with their characteristic structural and biomechanical anisotropies, Aim 2 - Engineer customized craniofacial tissues using an advanced bioreactor-imaging platform, and Aim 3 - Evaluate the tissue engineering approach to craniofacial reconstruction in a large animal model (Yucatan pig).